Then, 100 μl of a streptavidin-alkaline phosphatase complex preparation (Mabtech) was added to each well at a dilution of 1 : 1000 and incubated for 1 h at room temperature.
SH carried out cardiomyocyte studies, SR preparation and phosphatase activity studies.
To inhibit phosphatase activity during the preparation of cell lysates, phosphatase inhibitors (1 mM Na3VO4, 1 mM β-Glycerophosphate, 2.5 mM Sodium Pyrophosphate, 1 mM Sodium Fluoride) were also included.
For each of the four extracts generated from sample TP a fraction was treated with DNA phosphatase and the template preparation procedure was performed using 80°C as the denaturation temperature.
In contrast, immunoblotting the same samples with the pS65-TFIIB antibody showed a significantly lower signal in the TFIIB immunoprecipitate derived from the preparation that lacked phosphatase inhibitors.
Immunoblotting with the general TFIIB antibody demonstrated equivalent immunoprecipitation of TFIIB regardless of the presence or absence of phosphatase inhibitors during the lysate preparation (quantitation is shown below the autoradiogram).
To eliminate or minimize artifacts during sample preparation, we inhibited proteases and phosphatases as described under 'additional phosphatase and protease inhibitors' in the Material and Methods section.
Western blots of phospholamban and calcium/calmodulin-dependent protein kinase II, and serine/threonine phosphatase activity were studied in heart preparations.
Controls were run using an identical template preparation procedure but with no phosphatase treatment.
The BsmBI-digested HA was ligated into a dephosphorylated (Antarctic Phosphatase, M0289L; New England Biolabs) and BsmBI-digested preparation of the bidirectional reverse-genetics plasmid pHW2000 (Hoffmann et al., 2000) using T4 DNA ligase (M0202S; New England Biolabs).
